Cytochrome P450 monooxygenases often determine the clearance of candidate drugs limiting their efficacy. These enzymes can also be sources of off-target toxicity. Our long term objective is to understand the structural determinants of substrate recognition by P450 enzymes. Collectively and individually the human drug metabolizing P450s can metabolize structurally diverse range of substrates, and this is likely to reflect in part the flexibility of each enzyme and structural adaptations for substrate binding. Ths underscores the necessity to determine multiple structures of individual enzymes in complex with structurally dissimilar drugs to understand the contribution of conformational flexibility to drug binding. Specific changes in the active site architectures of P450s 1A2, 2C19 and 3A5 induced by interaction with chemically diverse substrates or inhibitors will be identified to delineate the range of adaptive changes that can occur for each enzyme. Studies of 2D6 will characterize the effects of genetic variation on substrate binding for the T108I and R296C variants that have high frequencies of occurrence and are known to alter the kinetic properties of the enzyme. Additionally, the active site topologies of P450s 2J2, 3A7 and representative family 4 enzymes will also be determined to identify structural characteristics that control the important functional contribution made by these enzymes to drug metabolism and the clearance of xenobiotics and excess endogenous compounds. Collectively, these studies will address significant gaps in our knowledge of P450 structure as it relates to function, and provide important information and tools for optimizing for lead compound optimization in drug development to improve efficacy and reduce risks of drug-drug metabolic interactions.